This invention relates to field of plant genetic engineering. In particular, it relates to promoters useful for inducible, transient expression of heterologous genes.
In the production of transgenic plants it is extremely valuable to have a variety of plant promoters that can be induced in response to various environmental stimuli. Currently, most inducible plant promoters are induced by application of chemicals. For example, plant promoters induced by tetracyline (Caddick et al. Nature Biotechnology 16:177-180 (1998)), dexamethasone (Gatz, Annu. Rev. Plant Physiol. Plant Mol. Bio. 48:89-108 (1997)) and ethanol (Aoyoma et al. Plant Journal 11:605-612 (1997)) are known. Identification of plant promoters induced by non-chemical stimuli would be extremely useful.
One of the most common plant responses to wounding or flooding is the induction of ethylene synthesis. Ethylene plays an important role in plant growth and development, including the control of seed germination, leaf senescence, floral fading, senescence and fruit ripening. Besides the stimulation by internal factors, ethylene synthesis is also stimulated by external factors. These include the induction of ethylene by chemicals and stresses, such as the attack by pathogens and environmental stresses, such as pollution, drought, chilling, heating, wounding and water logging, i.e. the soil flooding of plants (Jackson, M. B. Annu. Rev.Plant Physiol., 36, 145-175 (1985); Abeles et al., Ethylene in Plant Biology, Ed. 2, Academic Press, San Diego (1994)).
Ethylene is synthesized from S-adenosylmethionine via 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene (Adams, D. Q. and Yang, S. F., Proc. Natl. Acad. Sci. USA, 76, 170-174 (1979)). The reactions are catalyzed by ACC synthase (ACS), the key enzyme of ethylene synthesis and ACC oxidase (ACO) (Kende H, Annu. Rev. Plant Physiol., 44, 283-307 (1993)).
Flooded tomato roots synthesize the ethylene precursor ACC which is transported via the xylem to the leaves within 6 to 12 hours (Bradford K J. and Yang S. F., Plant Physiol., 65, 322-326 (1980)). The ethylene produced by waterlogged plants can be interpreted as an early warning of deteriorating soil conditions, inducing changes above ground to increase stress tolerance. Anaerobiotic conditions caused by flooding induce transcription of ACC synthase mRNAs in roots (Zarembinski T. I. and Theologis A., Mol. Biol. Cell 4, 363-373 (1993); Olson et al., J. Biol. Chem., 270, 14056-14061 (1995); Zarembinski, T. I. and Theologis, A. Plant Mol. Biol., 33, 71-77 (1997)). ACC synthase is encoded by a multigene family and in the tomato seven members of this gene family, LE-ACS1a, 1b, 2, 3, 4, 5 and 6 are known and differentially expressed (Van der Straeten et al., Proc.Natl.Acad.Sci. USA, 87,4859-4863 (1990); Rottmann et al., J.Mol.Biol., 222, 937-961 (1991); Olson et al., Proc. Nat. Acad. Sci. USA 88, 5340-5344 (1991); Yip et al., Proc. Natl. Acad. Sci. USA, 89, 2475-2479 (1992); Oetiker et al., Plant Mol. Biol., 34, 275-286 (1997)).
The gene LE-ACS2, commonly known as the ACC synthase that causes fruit ripening is induced in flooded roots (Van der Straeten et al., Proc.Natl.Acad.Sci. USA, 87,4859-4863 (1990); Rottmann et al., J.Mol.Biol., 222, 937-961 (1991); Parsons B. L. and Mattoo A. K., Plant Mol. Biol., 17, 453-464 (1991); Oeller, Science, 254, 437-439 (1991); Olson et al., J. Biol. Chem., 270, 14056-14061 (1995)). However, LE-ACS2 cannot be solely responsible for flooding induced ethylene because ACC is delivered from root to shoot within 6 to 12 hours after flooding but the LE-ACS2 transcript appears only 8 hours after flooding of the roots (Bradford K J. and Yang S. F., Plant Physiol., 65, 322-326 (1980); English, et al., J. Exp. Botany 45, 33.-33. (1995); English et al., Plant Physiol., 109, 1435-1440 (1995))(Olson et al., J. Biol. Chem., 270, 14056-14061 (1995)). LE-ACS3 is also induced in flooded tomato roots, but half of its polyadenylated mRNA remains unspliced, a commonly observed phenomenon under anaerobiotic stress (Olson et al., J. Biol. Chem., 270, 14056-14061 (1995)). Such stress-induced splicing failures will not lead to a functional ACS protein and therefore, we were looking for further ACS transcripts, potentially responsible for the initial burst of ethylene produced by leaves of waterlogged plants.
Identification of plant genes induced by flooding or other environmental stresses would be useful in isolation and preparation of inducible plant promoters. For example, identification of new inducible promoters that are induced quickly and provide transient expression of heterologous nucleic acids would extremely useful in controlling plant responses to various environmental stimuli. The present invention addresses these and other needs.
The present invention provides recombinant expression cassettes comprising an ACS7 promoter operably linked to a heterologous polynucleotide sequence. The promoter is at least about 70% identical to SEQ ID NO:1, the LE-ACS7 promoter from tomato. The heterologous polynucleotide sequence is not critical part of the invention. In some embodiments, the polynucleotide encodes a polypeptide, for example, one that confers resistance to a plant pathogen. Alternatively, the heterologous polynucleotide can be operably linked to the ACS7 promoter in the antisense orientation.
The recombinant expression cassettes of the invention are typically incorporated into vectors suitable for production of transgenic plants. The vector will usually further comprise an independent terminator sequence, replication sequences and a selection marker sequence. The expression cassettes of the invention can also be incorporated into the genome of transgenic plant. Thus, the invention also provides transgenic plants comprising the recombinant expression cassette of the invention. The transgenic plant can be any species, for example, tomato.
The invention also provides methods of expressing a nucleic acid in a plant. The methods comprise providing a transgenic plant comprising a recombinant expression cassette of the invention, and, subjecting the plant to an environmental stimulus which activates the ACS7 promoter, thereby transcribing the heterologous nucleic acid. The environmental stimulus is typically flooding or wounding.
Definitions
The term xe2x80x9cstressxe2x80x9d refers to harm or damage done to organs and/or tissues of a plant, including the roots, stems, leaves, buds, etc. The cause of stress can be exposure of the plant to anaerobic conditions (e.g., as a result of flooding), wounding, (e.g., cutting, scraping, freezing, and pinching), exposure to chemicals, (e.g. herbicides, fungicides, and insecticides), exposure to pathogens, (e.g., bacteria, viruses, and fungi), or exposure to pests, (e.g. insects, and nematodes), and the like.
The term xe2x80x9cpromoterxe2x80x9d refers to regions or sequence located upstream and/or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A xe2x80x9cplant promoterxe2x80x9d is a promoter capable of initiating transcription in plant cells. Such a promoter can be derived from plant genes or from other organisms, such as viruses capable of infecting plant cells.
An xe2x80x9cACS7 promoterxe2x80x9d refers to a plant promoter capable of initiating transcription from an operably linked heterologous sequence and which is at least substantially identical to the promoter sequence in SEQ ID NO:1 (LE-ACS7, from tomato). The promoters of the invention need not be full length, the promoters are typically from about 1000 to about 2500 nucleotides in length, usually from about 1500 to about 2000 nucleotides and often between about 2300 and about 2450 nucleotides.
A polynucleotide sequence is xe2x80x9cheterologous toxe2x80x9d a second polynucleotide sequence if it originates from a different gene, or, if from the same gene, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a gene different from that from which the promoter was derived, or, if from the same gene, a coding sequence which is not naturally *associated with the promoter (e.g. a genetically engineered coding sequence or an allele from a different ecotype or variety).
The phrase xe2x80x9coperably linkedxe2x80x9d refers to functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates transcription of RNA corresponding to the second sequence.
The phrase xe2x80x9cnucleic acid sequencexe2x80x9d refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5xe2x80x2 to the 3xe2x80x2 end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role.
The term xe2x80x9cplantxe2x80x9d includes whole plants, shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g. vascular tissue, ground tissue, and the like) and cells (e.g. guard cells, egg cells, trichomes and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous.
A polynucleotide xe2x80x9cexogenous toxe2x80x9d an individual plant is a polynucleotide which is introduced into the plant by any means other than by a sexual cross. Examples of means by which this can be accomplished are described below, and include Agrobacterium-mediated transformation, biolistic methods, electroporation, and the like. Such a plant containing the exogenous nucleic acid is referred to here as a T1 (e.g. in Arabidopsis by vacuum infiltration) or R0 (for plants regenerated from transformed cells in vitro) generation transgenic plant. Transgenic plants that arise from sexual cross or by selfing are descendants of such a plant.
xe2x80x9cRecombinantxe2x80x9d refers to a human manipulated polynucleotide or a copy or complement of a human manipulated polynucleotide. For instance, a recombinant expression cassette comprising a promoter operably linked to a second polynucleotide may include a promoter that is heterologous to the second polynucleotide as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloningxe2x80x94A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley and Sons, Inc. (1994-1998)) of an isolated nucleic acid comprising the expression cassette. In another example, a recombinant expression cassette may comprise polynucleotides combined in such a way that the polynucleotides are extremely unlikely to be found in nature. For instance, human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second polynucleotide. One of skill will recognize that polynucleotides can be manipulated in many ways and are not limited to the examples above.
Two nucleic acid sequences or polypeptides are said to be xe2x80x9cidenticalxe2x80x9d if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms xe2x80x9cidenticalxe2x80x9d or percent xe2x80x9cidentity,xe2x80x9d in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers and Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
The phrase xe2x80x9csubstantially identical,xe2x80x9d in the context of two nucleic acids or polypeptides, refers to sequences or subsequences that have at least 60%, preferably 70%, more preferably 80%, most preferably 90-95% nucleotide or amino acid residue identity when aligned for maximum correspondence over a comparison window as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence.
One of skill in the art will recognize that two polypeptides can also be xe2x80x9csubstantially identicalxe2x80x9d if the two polypeptides are immunologically similar. Thus, overall protein structure may be similar while the primary structure of the two polypeptides display significant variation. Therefore a method to measure whether two polypeptides are substantially identical involves measuring the binding of monoclonal or polyclonal antibodies to each polypeptide. Two polypeptides are substantially identical if the antibodies specific for a first polypeptide bind to a second polypeptide with an affinity of at least one third of the affinity for the first polypeptide.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat""l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley and Sons, Inc., (1995 Supplement) (Ausubel)).
Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always  greater than 0) and N (penalty score for mismatching residues; always  less than 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=xe2x88x924, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat""l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.
xe2x80x9cConservatively modified variantsxe2x80x9d applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are xe2x80x9csilent variations,xe2x80x9d which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, in a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a xe2x80x9cconservatively modified variantxe2x80x9d where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
The following six groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (see, e.g., Creighton, Proteins (1984)).
An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
The phrase xe2x80x9cselectively (or specifically) hybridizes toxe2x80x9d refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
The phrase xe2x80x9cstringent hybridization conditionsxe2x80x9d refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biologyxe2x80x94Hybridization with Nucleic Probes, xe2x80x9cOverview of principles of hybridization and the strategy of nucleic acid assaysxe2x80x9d (1993). Generally, highly stringent conditions are selected to be about 5-10xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. Lower stringency conditions are generally selected to be about 15-30xc2x0 C. below the Tm. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30xc2x0 C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60xc2x0 C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 time background hybridization.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
In the present invention, genomic DNA or cDNA comprising nucleic acids of the invention can be identified in standard Southern blots under stringent conditions using the nucleic acid sequences disclosed here. For the purposes of this disclosure, suitable stringent conditions for such hybridizations are those which include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37xc2x0 C., and at least one wash in 0.2xc3x97SSC at a temperature of at least about 50xc2x0 C., usually about 55xc2x0 C. to about 60xc2x0 C., for 20 minutes, or equivalent conditions. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary xe2x80x9cmoderately stringent hybridization conditionsxe2x80x9d include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37xc2x0 C., and a wash in 1xc3x97SSC at 45xc2x0 C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
A further indication that two polynucleotides are substantially identical is if the reference sequence, amplified by a pair of oligonucleotide primers, can then be used as a probe under stringent hybridization conditions to isolate the test sequence from a cDNA or genomic library, or to identify the test sequence in, e.g., an RNA gel or DNA gel blot hybridization analysis.